This application relates generally to polyhalogenated compounds for use in contrast imaging, and more particularly to an oil-in-water emulsion containing such compounds for use as a tissue-specific contrast agent and/or delivery vehicle for therapeutic agents incorporated therein.
Imaging agents are used for diagnostic modalities, such as computed tomography (CT), magnetic resonance (MR), ultrasound or nuclear medicine, to enhance the image contrast between tissue types. It is a shortcoming in the present state of the art that most of the currently used imaging agents are limited in action to the vascular and/or extracellular compartments. Thus, every tissue that receives a normal blood supply will also receive the diagnostic agent. Tissue-specific image enhancement is therefore compromised. Non-specific agents that reside in the extracellular space are useful primarily to discriminate anatomical features of tissues and structures. However, an imaging agent that can deliver a diagnostic agent to the intracellular environment of a targeted tissue could provide a means of assessing the metabolic and/or biochemical activity of the targeted tissue in addition to providing the standard anatomical visualization achieved with extracellular imaging agents.
In addition to the foregoing, agents that localize in the extracellular spaces are cleared very rapidly from the body. Due to the limitations of imaging hardware, the minimum period of time required to collect the data used to form a diagnostic image is predetermined. Consequently, a contrast agent that clears too quickly from the body must be administered in a very large dose in order to maintain a concentration gradient sufficient to achieve an acceptable quality of the image. Thus, use of many currently available diagnostic imaging agents is a complicated process balancing the benefits of image enhancement against the dangers of injecting large volumes of material into a living being in a short period of time. In the case of CT imaging, diagnostic imaging agents are commonly administered to the patient in volumes as large as 150 to 250 ml at rates of 1.5 to 2.5 ml/sec. Injection of currently available agents at this rate can induce nausea, headaches convulsions and other undesirable and dangerous side effects. There is thus a need for a tissue-specific delivery vehicle that concentrates the imaging agent in a single targeted organ or tissue type and thus permits slower, controlled injection of a substantially smaller dose. Of course, a lower dose would also minimize the; potential for toxicity and side effects, as well as preclude the need for expensive: power injectors.
For therapeutic purposes, such as the delivery of radioactive therapeutic agents, would be advantageous to target specific tissue and reduce the destructive effects of the radioactive agents on surrounding tissue.
Some known strategies for achieving tissue-specific delivery include the use of vehicles such as liposomes, antibody-linked conjugates, and carbohydrate derivatives of the targeting compound. However, many of these known vehicles cannot form acceptable complexes with the moiety to be delivered, or fail to accumulate the complexed moiety in the target tissue in quantities sufficient to be effective for imaging and/or therapy.
One of the most accurate, non-invasive radiologic examination techniques available for detection of hepatic masses is CT using water soluble, urographic contrast agents. However, the contrast agents commonly used in this well-known technique suffer from the typical limitations that plague other known contrast agents, including, for example, the requirement that large doses be administered, a nonspecific biodistribution, and an extremely short (&lt;2 min) residence time in the liver. As a result, CT has not been consistently successful at detecting lesions smaller than about 2 cm in diameter. These significant limitations of the known agents preclude early detection and therapy of cancer, since many metastases are smaller than the detection limits of this CT technique.
The inability of water-soluble urographic agents to detect lesions less than 2 cm in diameter with acceptable consistency may be due, in part, to the rapid diffusion of these agents out of the vasculature into interstitial spaces, resulting in a rapid loss of contrast differential between normal liver tissue and tumors. There is, therefore, a need for a diagnostic contrast agent, or vehicle therefor, that delivers the contrast agent to the intracellular space of specific targeted tissue, such as liver tissue, so as to enhance the degree of selective visualization and further improve the detection limits of CT. A number of alternatives to water-soluble contrast agents have been investigated as potential liver CT contrast agents including, for example, radiopaque liposomes, iodinated starch particles, perfluoroctylbromide, iodipamide ethyl esters, and ethiodized oil emulsion (EOE-13). All of these agents are particulate in nature and of such a size that liver specificity is mediated primarily via sequestration by the reticulendothelial system (RES).
Liposomes, which are artificially prepared lipid vesicles formed by single or multiple lipid bilayers enclosing aqueous compartments, are particulate in nature, and hence, have potential for delivering agents contained therein to the RES. Investigators have attempted to load liposomes with both ionic and non-ionic water-soluble urographic or hepatobiliary contrast agents, or to incorporate brominated phosphatidylcholine into the bilayer membrane of the liposomes. However, stabilization of the resulting liposome against loss of contrast media from the bilayers has proven to be a major problem. Moreover, incorporation of neutral lipophilic agents into the bilayer is limited by the low solubility of the lipophilic agents in the membrane matrix and the restricted loading capacity of the liposome.
Several monobrominated perfluorocarbons have been evaluated as contrast agents in animals. The most common of these, perfluoroctylbromide, has been shown to concentrate in the reticuloendothelial cells of the liver, spleen, and other organs. The long residency times (weeks to months) and the large doses (5-10 g/kg) necessary for suitable opacification will most likely preclude the use of monobrominated perfluorocarbons in humans for diagnostic imaging purposes, unless a means of specifically delivering small doses to a targeted organ is developed.
The most promising of the investigational agents mentioned above, EOE-13, has been extensively studied in both animals and humans in the United States. EOE-13, an emulsion of iodinated poppy seed oil (37% iodine by weight) in saline, offered considerable improvement in the detection of space-occupying lesions in the liver and spleen as compared to conventional water-soluble urographic agents. Despite acceptable clinical diagnostic efficacy, a high incidence of adverse reactions, including fever, chills, thrombocytopenia, hypotension, and respiratory distress, was reported. Moreover, additional problems were encountered in the sterilization of the EOE-13 preparation. These problems led to discontinuation of the use of EOE-13 in humans.
Recently, investigators have demonstrated a direct relationship between the size of emulsion particles and involvement of macrophages. In this study, the investigators tested three iodinated lipid emulsions, including EOE-13, having mean particle diameters ranging from 400-2000 nm. They observed a marked swelling of Kupffer cells which, when coupled with sinusoidal endothelial damage, resulted in sinusoidal congestion. Sinusoidal congestion often activates macrophages, resulting in the release of toxic mediators which may be responsible, in part, for the adverse reactions seen with these relatively large-sized particulate, preparations. As a result, the investigators emulsified iodinated ethyl esters of fatty acids derived from poppyseed oil (Lipiodol.TM.-UF, Laboratoire Guerbet, France) with egg yolk phospholipids, in order to provide a preparation of smaller, more uniform particle size, called Intraiodol (not commercially available; see, for example, Acta Radiologica, Vol. 30, pages 407-412 and 449-457, 1989),. Intraiodol.TM. emulsion has a particle size range of 100 to 650 nm (distribution means diameter 310 nm). Initial results obtained with Intraiodol in animals and humans demonstrated a significant reduction in adverse reactions relative to those observed with EOE-13. However, Intraiodol.TM. emulsion continues to suffer from many disadvantages common to other prior art iodinated lipid emulsion contrast agents, including failure to achieve true specificity due to inter alia, size and contamination of the liposome composition with foreign particulates, resulting in two disadvantages: 1) delivery to the RES, and 2) inability to achieve shelf and heat stability. Moreover, in this prior art compound, the iodine necessary for CT opacification is attached in an aliphatic linkage, which is well known to exhibit diminished in vivo stability.
Although Intraiodol.TM. emulsion, and other similar oil-in-water emulsions, have been called "chylomicron remnant-like" and "hepatocyte specific," these agents located significantly in the spleen, which does not contain hepatocytes. More particularly, a true hepatocyte-specific contrast agent will not locate substantially in the spleen, which does not contain hepatocytes. A true hepatocyte-specific contrast agent will not locate substantially in the spleen and other RES associated organs, unless there is saturation of the initial receptor-mediated process, so that there is a shift in delivery to the cells of the RES. Further, a true hepatocyte-specific agent is cleared primarily through the biliary system. None of the aforementioned emulsions has demonstrated these characteristics of hepatocyte-specificity in biliary clearance studies.
To summarize briefly the natural lipid transport system, lipids are transported in the plasma mainly in the form of free fatty acids, triglycerides, and cholesteryl esters. Free fatty acids are transported as a complex with plasma albumin, whereas triglycerides and cholesteryl esters are transported in the lipophilic core of plasma lipoprotein. The surface of a plasma lipoprotein membrane comprises a monolayer of phospholipid, cholesterol, and specific proteins known as apolipoproteins which regulate the entry and exit of particular lipids at specific targets. Cholesterol and triglycerides from dietary sources are absorbed by the intestinal tract and incorporated into chylomicrons, which are subsequently secreted into and transported through the thoracic duct until they reach the circulation. Once in circulation, there is a rapid transfer of apoprotein C-II from circulating high density lipoprotein to the chylomicrons. Once associated with apoprotein C-II, the chylomicrons are acted upon by lipoprotein lipase in the capillary beds of several peripheral tissues including adipose, muscle (skeletal and heart), and lung. Lipoprotein lipase hydrolyzes much of the core triglyceride to glycerol and free fatty acids, most of which are taken up by the tissues for storage or oxidation. The remaining triglyceride-depleted chylomicrons, called chylomicron remnants, now contain lesser amounts of triglycerides, with cholesteryl esters as the main lipid component and apoprotein B (Apo B) and apoprotein E (Apo E) as the major apoprotein components. Chylomicron remnants are cleared very rapidly from the circulation by the liver via a receptor-mediated process that recognizes Apo B and Apo E. While lipoprotein lipase is the enzyme responsible for the hydrolysis of plasma triglycerides in extrahepatic tissues, hepatic triglyceride lipase is implicated in hepatic triglyceride hydrolysis and remnant uptake by hepatocytes. Hepatic clearance of chylomicron remnants from the circulation occurs mainly via the hepatocytes (parenchymal cells) rather than Kupffer cells (nonparenchymal cells).
Radiopaque lipids are generally incorporated into such emulsions to create radiopaque embodiments of such contrast-producing oil-in-water emulsions. In iodinated embodiments, iodine-containing lipids known in the art include iodinated fatty acids in the form of glycerol or alkyl esters. However, the iodine-containing lipids are preferably synthetic aromatic compounds of known purity that are stabilized against in vivo degradation of the iodine linkage. Illustrative examples of radioactive or non-radioactive halogenated triglycerides useful in the practice of the invention include, without limitation, iodinated triglycerides of the type described in U.S. Pat. No. 4,73,075 issued on Oct. 10, 1989; U.S. Pat. No. 4,957,719 issued on Sept. 18, 1990; and U.S. Pat. No. 5,093,043 issued on Mar. 3, 1992. Exemplary iodinated triglycerides are 2-oleoylglycerol-1,3-bis[7-(3-amino-2,4,6-triodophenyl)heptanoate] (DHOG) and 2-oleoylglycerol-1,3-bis[4-(3-amino-2,4,6-triiodophenyl)butanoate] (DBOG).
However, these compounds are complicated and, hence, expensive to manufacture. Moreover, because such compounds generally require an oil carrier, it is difficult to sufficiently concentrate the radiopaque substance to accomplish certain diagnostic and/or therapeutic ends requiring a high concentration of the; radioisotope.
Accordingly, there remains a great need in the art for target-specific delivery vehicles or compositions, including contrast-producing oils and oil-in-water emulsions, for delivery of diagnostic, therapeutic, and other biologically active or inactive agents, particularly for hepatocyte specific contrast-producing oils and oil-in-water emulsions for delivery of diagnostic or therapeutic agents, such as radioisotopes, to the liver. There also remains a need in the art for low cost low molecular weight radiopaque contrast agents that are lipophilic in character for inclusion in such oil-in-water emulsions for delivery to target specific sites.
It is, therefore, an object of this invention to provide an improved oily or lipophilic radiopaque contrast agent that is inexpensive to manufacture and can be used in an oil-in-water emulsion or without an oil carrier as a target-specific delivery vehicle:, such as an hepatocyte selective delivery vehicle.
It is a further object of this invention to provide a delivery vehicle, specifically a target-selective oil-in-water emulsion for delivery of lipophilic agents, or lipophilic derivatives of water soluble agents, such as contrast agents, to the intracellular spaces of the targeted tissue.
It is another object of this invention to provide a target-specific delivery vehicle, specifically a hepatocyte-selective oil-in-water emulsion, which is chylomicron remnant-like with respect to size and biodistribution characteristics.
It is also an object of this invention to provide a target-selective oil-in-water emulsion that is shelf stable and heat stable so that it can be heat sterilized.
It is a further object of this invention to provide a method of preparing a target-selective oil-in-water emulsion which is chylomicron remnant-like, shelf and heat stable, and substantially free of liposome contamination.